Project Summary The histone deacetylase (HDAC)-containing Sin3 complex is a chromatin modifier crucial for the regulation of gene expression. Inhibitors against HDACs are currently in use for treatment against cancer, while haploinsufficiency of SIN3A has been linked to neurological development disorders. The Sin3/HDAC complex is composed of a core group of subunits; however, our data and others have identified additional peripheral subunits that interact with the core in a mutually exclusive manner. Furthermore, these peripheral subunits consist of a series of paralogues, each with documented links to human diseases like cancer. These results indicate that there likely exist many distinct Sin3 complexes that have yet to be defined. The possible combinations of paralogues that appear with the core complex are difficult to discern with traditional proteomic analysis, which typically provides a list of proteins enriched in samples compared to controls. Therefore, there is a critical need for strategies that are capable of discerning between heterogeneous protein complexes. Here, my goal is to determine the combinations of paralogues that interact with the core complex in their native cellular environment. I will accomplish this goal by pursuing two specific aims. In the first specific aim, I will identify direct interactions in the Sin3/HDAC protein interaction network using biochemical approaches and quantitative imaging techniques in live cells. From this information, we will be able to build the first model of the Sin3 protein interaction network where direct interactions are defined in a systematic manner. In the second specific aim, I will develop a novel dual-tagged system to investigate paralogue specific interactions in live cells using quantitative proteomic and quantitative imaging techniques. From this aim, I will develop a new approach for studying protein complexes when two proteins are likely competing for the same interactions in the same cells. The successful completion of this project will significantly expand our understanding of the diversity of Sin3 complexes in mammalian cells, which has broad implications for chromatin remodeling in normal and diseased cellular states.